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Opsonic phagocytosis of Plasmodium falciparum merozoites: mechanism in human immunity and a correlate of protection against malaria BMC Medicine 2014, 12:108
doi:10.1186/1741-7015-12-108
Faith HA Osier (
[email protected]) Gaoqian Feng (
[email protected]) Michelle J Boyle (
[email protected]) Christine Langer (
[email protected]) Jingling Zhou (
[email protected]) Jack S Richards (
[email protected]) Fiona J McCallum (
[email protected]) Linda Reiling (
[email protected]) Anthony Jaworowski (
[email protected]) Robin F Anders (
[email protected]) Kevin Marsh (
[email protected]) James G Beeson (
[email protected])
ISSN Article type
1741-7015 Research article
Submission date
16 March 2014
Acceptance date
6 June 2014
Publication date
1 July 2014
Article URL
http://www.biomedcentral.com/1741-7015/12/108
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Opsonic phagocytosis of Plasmodium falciparum merozoites: mechanism in human immunity and a correlate of protection against malaria Faith HA Osier1,2,† Email:
[email protected] Gaoqian Feng2,† Email:
[email protected] Michelle J Boyle2 Email:
[email protected] Christine Langer2 Email:
[email protected] Jingling Zhou2 Email:
[email protected] Jack S Richards2 Email:
[email protected] Fiona J McCallum3 Email:
[email protected] Linda Reiling2 Email:
[email protected] Anthony Jaworowski2 Email:
[email protected] Robin F Anders4 Email:
[email protected] Kevin Marsh1 Email:
[email protected] James G Beeson2,5,* Email:
[email protected] 1
KEMRI Centre for Geographic Medicine Research-Coast, Kilifi, Kenya
2
Centre for Biomedical Research, The Burnet Institute, 85 Commercial Road, 3004 Melbourne, Victoria, Australia 3
Australian Army Malaria Institute, Enoggera, Queensland, Australia
4
Department of Biochemistry, La Trobe University, Melbourne, Australia
5
Department of Microbiology, Monash University, Wellington Rd, Clayton, VIC 3800, Australia *
Corresponding author. Department of Microbiology, Monash University, Wellington Rd, Clayton, VIC 3800, Australia †
Equal contributors.
Abstract Background An understanding of the mechanisms mediating protective immunity against malaria in humans is currently lacking, but critically important to advance the development of highly efficacious vaccines. Antibodies play a key role in acquired immunity, but the functional basis for their protective effect remains unclear. Furthermore, there is a strong need for immune correlates of protection against malaria to guide vaccine development.
Methods Using a validated assay to measure opsonic phagocytosis of Plasmodium falciparum merozoites, we investigated the potential role of this functional activity in human immunity against clinical episodes of malaria in two independent cohorts (n = 109 and n = 287) experiencing differing levels of malaria transmission and evaluated its potential as a correlate of protection.
Results Antibodies promoting opsonic phagocytosis of merozoites were cytophilic immunoglobulins (IgG1 and IgG3), induced monocyte activation and production of pro-inflammatory cytokines, and were directed against major merozoite surface proteins (MSPs). Consistent with protective immunity in humans, opsonizing antibodies were acquired with increasing age and malaria exposure, were boosted on re-infection, and levels were related to malaria transmission intensity. Opsonic phagocytosis was strongly associated with a reduced risk of clinical malaria in longitudinal studies in children with current or recent infections. In contrast, antibodies to the merozoite surface in standard immunoassays, or growth-inhibitory antibodies, were not significantly associated with protection. In multivariate analyses including several antibody responses, opsonic phagocytosis remained significantly associated with protection against malaria, highlighting its potential as a correlate of immunity. Furthermore, we demonstrate that human antibodies against MSP2 and MSP3 that are strongly associated with protection in this population are effective in opsonic phagocytosis of merozoites, providing a functional link between these antigen-specific responses and protection for the first time.
Conclusions Opsonic phagocytosis of merozoites appears to be an important mechanism contributing to protective immunity in humans. The opsonic phagocytosis assay appears to be a strong correlate of protection against malaria, a valuable biomarker of immunity, and provides a
much-needed new tool for assessing responses to blood-stage malaria vaccines and measuring immunity in populations.
Keywords Malaria, merozoites, monocytes, phagocytosis, immunity, vaccines, Plasmodium falciparum
Background Knowledge of the mechanisms that mediate protective immunity against Plasmodium falciparum malaria in humans is currently very limited, and this has been a major barrier to vaccine development. In malaria-endemic areas the severity and frequency of clinical malaria declines with increasing age and repeated exposure to infections, reflecting the acquisition of specific immunity [1]. Antibodies are known to be key components of naturally-acquired immunity against P. falciparum [1,2], and passive transfer of immunoglobulins from immune donors to individuals with P. falciparum infection has been shown to reduce parasitemia and clinical symptoms [3,4]. Merozoites are a major target of these acquired antibody responses [5-8]. However, the mechanisms mediating protective humoral immunity and the key targets of functional antibodies remain poorly understood. In addition, there is a lack of strong immune correlates of protective immunity for use in vaccine development and population monitoring in malaria control programs [9]. The growth inhibition assay (GIA) is the only widely used functional assay in studies of acquired human immunity and candidate bloodstage vaccines [10]. However, associations between growth-inhibitory antibodies and protective immunity have been weak and inconsistent [10-13], implying that other mechanisms are important, but these remain undefined. Recently, a neutrophil-based antibody-dependent respiratory burst (ADRB) assay was shown to be a correlate of acquired immunity in two endemic populations in Senegal, but these findings have not as yet been reproduced in other cohort studies in Africa [14]. We investigated the role of antibody-mediated opsonic phagocytosis of P. falciparum merozoites by monocytes. Human antibodies to P. falciparum merozoites are predominantly of the cytophilic (IgG1 and IgG3) sub-types that interact with monocytes and other cells, via Fc-gamma receptors [8,15-17]. In addition to direct clearance of merozoites, opsonic phagocytosis by monocytes may also stimulate the release of cytokines or other mediators that subsequently promote parasite killing [18]. Although prior studies have shown that antibodies can promote phagocytosis of merozoites [19-21], how these antibodies are acquired and/or boosted is unknown, and their targets and relationships to other immune measures have not been defined. There are no longitudinal studies of these responses in African children who are at the greatest risk of malaria or studies to define how they are acquired and/or boosted. We developed and validated an efficient assay with good throughput to measure antibodymediated opsonic phagocytosis of merozoites using the human monocytic THP-1 cell line. We studied the properties of opsonic phagocytosis antibodies, identified merozoite target antigens and demonstrated resultant monocyte activation. In detailed longitudinal studies of African children we defined the acquisition of opsonic phagocytosis antibodies, and show that it is strongly associated with protection against malaria, suggesting opsonic phagocytosis antibodies could be used as a valuable correlate of protection in malaria vaccine development.
Methods Study population Two different longitudinal cohort studies, referred to as Chonyi and Ngerenya, were included to address different aspects of the acquisition and boosting of antibodies, their relationship to immunity, and the impact of malaria transmission rates on antibodies. Details of the study area and population are published [22]. Malaria transmission occurs in two seasonal peaks, with average annual entomological inoculation rates (EIRs) of 20 to 53 (Chonyi), and 10 (Ngerenya) infective bites/person/year [23,24]. Briefly, participants were recruited during cross-sectional surveys in October 2000 (Chonyi, n = 536) and October 2002 (Ngerenya, (n = 295)), at the start of a malaria transmission season. A single blood sample was collected at recruitment and participants were subsequently monitored actively each week for six months to detect clinical episodes of malaria. Regular screening for parasitemia was not performed during the follow up visits, but was measured only when participants reported any symptoms suggestive of malaria. Local age-specific criteria defining clinical episodes of malaria were: fever (>37.5°C) plus any parasitemia for children less than one year old, and fever plus a parasitemia >2,500/µL for participants older than one year [22]. In Ngerenya, children also underwent active surveillance for malaria in the six months prior to sample collection. In this report, data are presented for all children from Ngerenya for whom a sample was available (n = 287) and the subset of children from Chonyi who were asymptomatically parasitized (parasite-positive) at the time of sampling (n = 109). For Chonyi, this subset was studied for two main reasons; firstly, although the original cohort was made up of children and adults, 90% of all clinical episodes observed during six months of monitoring occurred in children ≤10 years old; therefore, adults were excluded from the analysis of antibodies in relation to the risk of clinical malaria. Secondly, protective associations have only been observed in the subset of children who were asymptomatically parasitized (parasite-positive) at recruitment and the incidence of malaria in those who were parasite negative at enrollment was low [6,25-30]. Therefore, this subset was considered ideal to test the hypothesis that the phagocytosis index was a correlate of protective immunity against clinical episodes of malaria, and comprised children up to ten years of age who were parasite positive at recruitment (n =109). For the initial evaluation of opsonic phagocytosis responses and validation of the assay, and for performing detailed comparisons between opsonic phagocytosis and other measures of malaria immunity, a random selection of samples from Ngerenya children and adults (n = 33) was used for which we had sufficient volumes of sera to perform multiple antibody testing. Total immunoglobulin G (IgG) was also purified from these samples for use in assays. Pooled serum from 20 adult blood donors from the same village was used to affinity-purify antigen-specific antibodies. A reference Malaria Immune Globulin (MIG) reagent (Central Laboratory of the Swiss Red Cross Blood Transfusion Service, Berne Switzerland) [31] was used for validation experiments and as a positive control for the cohort assays. This preparation contains 50 mg/ml of immunoglobulins (98% IgG) purified from a pool of healthy Malawian adult plasma and was originally manufactured to test its potential use as an adjunct therapy to quinine in the treatment of cerebral malaria. Written informed consent was obtained from all study participants or their parents/guardians. Ethical approval was granted by the Kenya National Research Ethics Review Committee (SSC No. 1131).
Laboratory methods Culture of THP1 cells THP1 cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 with 0.002 mol/L L-glutamine, 1.5 g/L sodium bicarbonate, 0.01 mol/L HEPES, 5 × 10−5 mol/L 2mercaptoethanol, and 10% fetal bovine serum [32]. Cell density was monitored closely and maintained between 1 x 105 and 1 x 106 cells/mL. Cells were passaged every six days, when cell density approached 1 × 106 cells/mL.
Culture of Plasmodium falciparum The laboratory-adapted P. falciparum line D10 was cultured in RPMI- N-2hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) with 0.5% Albumax and 0.18% NaHCO3 [33]. Cultures were maintained below 10% parasitemia and synchronized by sorbitol treatment.
Isolation of free merozoites Merozoites were isolated directly from culture using previously published methods [34,35]. Briefly, late stage pigmented trophozoites were harvested by magnetic purification on columns and then cultured in medium supplemented with the protease inhibitor transepoxysuccinyl-L-leucylamido(4-guanidino) butane (E64) for eight hours to allow maturation to schizonts without rupture. Mature schizonts were collected and passed through a 1.2 µm filter to release and purify merozoites. Free merozoites were stained with ethidium bromide (EtBr) at a final concentration of 1 µg/mL for 30 minutes followed by three washes in RPMI. The cell density was determined using relative counting against CountBright™ Absolute Counting Beads (Invitrogen, Mount Waverly, Victoria, Australia) on a BD FACSCalibur (BD Biosciences, North Ryde, New South Wales, Australia) flow cytometer. The merozoites were then resuspended at 5 × 107 merozoites/mL in RPMI-HEPES and used in assays as described.
Isolation of human peripheral blood mononuclear cells Human peripheral blood was collected into ethylenediaminetetraacetic acid (EDTA)-coated vacutainers from malaria naïve donors. Whole blood was diluted with an equal volume of PBS and overlayed on 15 ml of Ficoll. The tube was centrifuged at 400 x g for 40 minutes before collection of the buffy coat. Cells were washed three times with PBS supplemented with newborn calf serum (NCS) and resuspended in RPMI-1640 supplemented with 10% human serum. The peripheral blood mononuclear cells (PBMCs) were then stored at 4°C until use.
Phagocytosis using undifferentiated THP-1 cells Our method was adapted from an established assay for the opsonic phagocytosis of P. falciparum-infected erythrocytes by undifferentiated THP-1 cells [36,37]. Briefly, freshly cultured THP-1 cells were counted and resuspended at a final concentration of 5 × 105/mL in THP-1 culture medium. Freshly isolated merozoites were transferred into 96-well Ubottomed plates (30 µL/well at 5 × 107 merozoites) that had been pre-coated with fetal calf serum (FCS) (200 µL of FCS, incubated for one hour, followed by a single wash with
incomplete RPMI). All antibodies used for opsonization were heat inactivated to exclude any influence of complement. For opsonization, 3.5 µL of test serum was incubated with 30 µL of merozoites (pre-stained with EtBr) for one hour at room temperature in the dark. The plate was washed three times using incomplete RPMI, before resuspension in 150 µL THP-1 culture medium. To obtain three replicates, 50 µL of the opsonized merozoites were coincubated with 100 uL each of THP-1 cells at 5 × 105 cells/mL in FCS at 37°C for 10 minutes for phagocytosis. Phagocytosis was stopped by the addition of 50 µL cold PBS supplemented with NCS. Plates were immediately washed to remove free or loosely attached merozoites. Three washes were performed using the same buffer at 4°C (centrifugation at 300 × g for four minutes). THP-1 cells were then fixed with 2% paraformaldehyde (PFA) for two hours before analysis by flow cytometry. Several controls were included for each assay: 1) non-opsonized merozoites; 2) merozoites opsonized with non-malaria exposed sera; and 3) merozoites opsonized with pooled highly reactive sera from adults exposed to malaria (MIG). Selected assays had an additional control in which THP-1 cells were pre-incubated with cytochalasin D to inhibit phagocytosis. Flow cytometry was performed in a 96-well format on a BD FACS CantoII (BD Biosciences). In preliminary experiments we established that a ratio of merozoites:THP-1 cells of 10:1 was optimal. The level of phagocytosis was determined by counting the percentage of THP-1 cells that had ingested merozoites and is referred to as the Phagocytosis Index (PI). Results are presented as a relative phagocytosis index (RPI%), with the PI being expressed as a ratio to that of a standard positive control run in each assay. Samples were considered positive for phagocytosis if the RPI exceeded the mean plus three standard deviations of a panel of 10 non-malaria exposed sera from Melbourne blood donors (Melbourne controls).
Phagocytosis using freshly isolated peripheral blood mononuclear cells Isolated human PBMC were resuspended in RPMI-1640 supplemented with 10% FBS at a final concentration of 5 × 106/mL. Freshly isolated merozoites were opsonized and stained with EtBr followed by co-incubation with PBMCs for 10 minutes. The monocyte population was gated on flow-cytometry dot plots using light scatter characteristics and the percentage of EtBr positive monocytes used to determine the phagocytosis index.
Immunofluorescence staining for monocyte activation markers Isolated PBMCs were resuspended in RPMI-1640 supplemented with 10% human serum at a concentration of 5 × 106/mL. PBMCs (100 µL) were added to polypropylene tubes and kept on ice. Freshly isolated merozoites were opsonized with either hyper-immune human IgG or serum from malaria-naïve Melbourne donors for one hour before being re-suspended in RPMI-1640 supplemented with 10% human serum at a concentration of 5 x 107/mL. Merozoites (10 µL) were added to the PBMCs and co-incubated at 37°C for six hours. Brefeldin A and Monensin were added to each tube at concentration of 1:1000 and 1:1500, respectively, prior to co-incubation. Cells were then washed with cold fluorescence-activated cell sorting (FACS) buffer after co-incubation and monocytes were labelled by staining with anti-CD14-APC antibodies. Cells were stained with anti-CD69-V450 to determine monocyte activation [38-40]. The cells were fixed overnight with BD FACS fix buffer (BD Biosciences) then permeabilized with BD Perm/wash buffer. Production of intracellular tumor necrosis factor- α (TNF-α) was detected by staining with anti-TNF-α -PE antibodies. Cells were re-suspended in BD Fix buffer following intracellular staining.
Scanning electron microscopy Square glass coverslips (22 mm) were prepared by smearing with a 0.1% solution of polyethyleneimine (PEI) and then dried. Cell samples were incubated on PEI-coated glass coverslips for half an hour. Following incubation, the excess sample was drained, and coverslips with adhered cells were immersed in 2% glutaraldehyde in PBS for one hour. Coverslips were then rinsed three times in PBS for 10 minutes each before being dehydrated in increasing concentrations of ethanol consisting of 10, 20, 40, 60, 80 and 100% ethanol in water for 10 minutes for each step. The coverslips were then dried in a Balzers CPD 030 Critical Point Dryer (Balzers Pfeiffer, Balzers, Liechtenstein) and mounted onto 25-mm aluminum stubs with double-sided carbon tabs and then gold-coated in a Dynavac ‘Xenosput’ magnetron sputter coater (Dynavac, Hingham MA, USA). The cells on coverslips were imaged with the Philips XL30 field emission scanning electron microscope (Philips, Eindhoven, Netherlands) at a voltage of 2 kV.
Fluorescence microscopy Merozoites were stained with Hoechst after opsonization with purified IgG from malariaexposed Kenyan adult sera after which they were co-incubated with THP-1 cells. The THP-1 cells were then resuspended in 2% PFA and mounted on a glass slide. The slides were immediately analyzed by Zeiss Cell Observer (North Ryde, New South Wales, Australia. using a 100x magnification object lens.
Affinity purification of human and rabbit antigen-specific anti-merozoite antibodies Human antibodies against the K1 allelic version of MSP3 [41] and the FC27 allele of MSP2 [42] of P. falciparum were affinity purified from a 50 mL pool of plasma taken from malaria semi-immune adults in Kenya (described under Study Populations, n = 20) by column chromatography (CNBr-activated SepharoseTM 4B, GE Healthcare), according to the manufacturer's instructions, and as previously described [43].
Whole merozoite ELISA Purified whole merozoites [34,35] were resuspended in PBS supplemented with a cocktail of protease inhibitors (Roche, Castle Hill NSW, Australia). Whole merozoites were then plated in NUNC MaxisorpTM plates at 100 µL per well and incubated at 37°C for two hours (or overnight at 4°C), followed by six washes with PBS. The plates were then blocked with 200 µL of 1% casein in PBS at 37°C for two hours followed by two washes with PBS. Serum samples were diluted at 1:1000 in PBS and 100 µL of each sample was added to the ELISA plates in duplicate and incubated at 37°C for one hour, followed by six washes in PBS. The plates were further incubated with 100 µL per well of horseradish peroxidase (HRP)conjugated goat anti-human IgG antibody diluted at 1:2500 in 0.1% PBS-casein at 37°C for one hour followed by six washes with PBS. For determination of IgG1 and IgG3 subclasses, peroxidase conjugated anti-human IgG1/IgG3 antibodies were diluted at 1:1000 in 0.1% PBS-casein. Finally, 100 µL of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was added to each well and incubated for 20 minutes for color development. The reaction was stopped by the addition of 100 µL 1% SDS solution. Color development was quantified at 405 nm. Pooled human IgG from malaria exposed Kenyan adults was used at
1:1000 dilution as a positive control and individual sera from malaria non-exposed Melbourne adults were used at 1:1000 dilution as negative controls.
Data analysis Data were analyzed using Prism 5 (GraphPad Software, Inc) and Stata 11 (StataCorp). For the Ngerenya cohort, the presence/absence of phagocytosis was analyzed in relation to single and multiple clinical episodes of malaria using the modified Poisson regression model [44]. Subgroup analyses were performed for children who had recent exposure to malaria (positive malaria slide) in the six months before samples were collected. In Chonyi, >94% of children were positive for phagocytosis, precluding the use of the presence/absence of phagocytosis to relate to malaria risk. However, the levels of phagocytosis were nearly normally distributed allowing us to define three levels of phagocytosis by tertiles: high, medium and low. Risk of malaria episodes was analyzed for each tertile using standard survival analysis techniques [17]. All analyses included age as a potential confounder.
Results Human antibodies promote phagocytosis of merozoites We developed and validated an assay to measure opsonic phagocytosis using the human monocyte THP-1 cell line and purified intact merozoites isolated using recently established methods [34,35]; we refer to these as opsonic phagocytosis assays (OPA). We demonstrated that antibody-mediated opsonic phagocytosis: (1) was specific to the IgG fraction of malariaexposed sera in a dose-dependent manner; (2) was inhibited by pre-treatment of THP-1 cells with cytochalasin D, which is a known inhibitor of macrophage and monocyte phagocytosis; (3) was comparably quantified using either THP-1 cells or freshly isolated human PBMCs; and (4) robustly measured the internalization of merozoites into phago-lysosomes, as demonstrated by staining merozoites with the pH-dependent dye pHrodoTM, which only becomes fluorescent in the acid environment of phago-lysosomes (Figure 1A-D). We found equivalent levels of phagocytosis using merozoites stained with pHrodoTM, compared to those stained with EtBr, indicating that our assay robustly quantified internalized merozoites and was not confounded by surface bound merozoites. Opsonic phagocytosis was optimally detected at 10 minutes [see Additional file 1: Figure S1], and our OPA gave results that were reproducible over a wide range of phagocytic activity [see Additional file 1: Figure S2]; we demonstrated activity using merozoites purified from several different P. falciparum isolates (isolates D10, 3D7, E8B, CS2, W2mef). Opsonic phagocytosis led to activation of monocytes demonstrated by increased intracellular TNF-α production, which is thought to play an important role in parasite clearance and immunity [18], and up-regulated CD69 expression (Figure 2A-B). Scanning electron microscopy captured striking images showing attachment of merozoites to THP-1 cells and the commencement of phagocytosis and internalization (Figure 3A). Internalized merozoites could also be clearly seen using immunofluorescence microscopy (Figure 3B).
Figure 1 Phagocytosis assay validity. (A) Phagocytosis of whole merozoites is specific to malaria-immune sera and efficiently inhibited by treatment with cytochalasin D. (B) The IgG fraction purified from serum mediates phagocytosis in a concentration dependent fashion, in assays using cultured THP-1 cells and freshly isolated PBMCs. (C) Flow cytometry histogram overlay contrasting the phagocytosis in monocytes from human PBMCs when freshly isolated merozoites are opsonized with purified IgG from malaria-immune adults (grey line) with unopsonized merozoites (black line). (D) Equivalent levels of phagocytosis obtained when merozoites were stained with the pH dependent dye pHrodoTM or with ethidium bromide, indicating internalization of merozoites into acidic phago-lysosomes. Experiments were conducted using malaria-immune IgG (MIG). PBMCs. peripheral blood mononuclear cells. Figure 2 Monocyte activation and cytokine production following phagocytosis of merozoites. Production of intracellular TNF-α (A) and surface expression of CD69 (B) was significantly elevated in the monocytes co-incubated with malaria-immune IgG (MIG) opsonized merozoites (black solid line), while that in monocytes co-incubated with merozoites opsonized with non-immune Melbourne sera (grey solid line) was no different from baseline levels detected before phagocytosis (dark dotted line). Figure 3 Visualization of opsonic phagocytosis of merozoites by THP-1 cells. (A) Scanning electron microscopy of THP-1 cells before and during ingestion of merozoites. (B) Immunofluorescence microscopy of phagocytosis of merozoites opsonized with purified IgG from malaria-immune adults (MIG). THP-1 cells were visualized by differential interference contrast (DIC), while merozoites were visualized with Hoechst (blue). Figures represent data acquired from at least two independent experiments.
Relationship to other measures of immunity Antibody responses were studied in detail in a subset of samples (n = 33) from children and adults to define relationships between opsonic phagocytosis antibodies and other measures of immunity. To understand better the antibodies promoting phagocytosis and the relationship between antibody binding to the surface of merozoites with OPA, we developed an assay to measure antibodies to intact merozoites by ELISA. Activity in OPA was significantly and positively correlated with IgG reactivity against intact purified merozoites, and mediated predominantly by IgG1 and IgG3 subclasses (Figure 4A-B). The GIA is currently the most widely used functional assay for anti-merozoite antibodies, but has not been consistently associated with protection in naturally-acquired or vaccine-induced immunity. We found that opsonic phagocytosis was only weakly correlated with the ability of the same purified IgG to inhibit parasite growth in a standard GIA (Spearman’s rho −0.358, P = 0.041, Figure 4C); similarly, total IgG against whole merozoites was also only weakly correlated with inhibitory activity in GIAs (Spearman’s rho −0.410, P = 0.018, Figure 4D). Others have reported variable correlations (negative, positive and non-significant) between growth inhibitory antibodies and exposure, or antibodies to merozoite surface proteins (MSPs), in some African populations [11,12,45], including Kenya, which has raised questions about the GIA as a valid correlate of human immunity.
Figure 4 Characteristics of antibodies promoting phagocytosis. (A) Pairwise correlation between the relative phagocytosis index and IgG ELISA optical density (OD) against whole merozoites. (B) IgG subclasses against whole merozoites measured by ELISA. (C) Pairwise correlation between the relative phagocytosis index and parasite growth inhibition measured in the GIA. (D) Pairwise correlation between IgG ELISA OD against whole merozoites and GIA. Data from GIAs (C and D) are expressed as parasite growth (%), relative to malarianaive controls. Experiments were conducted using purified IgG from adults and children in Ngerenya , n = 33. GIA, growth inhibition assay.
Acquisition of human antibodies promoting opsonic phagocytosis We next measured opsonic phagocytosis activity in samples from two separate longitudinal cohort studies of children performed in two sites with different levels of malaria transmission. Transmission intensity was low in Ngerenya and samples were available for 287 children, 0.1- to 8-years old, 20 (7%) of whom had asymptomatic P. falciparum infections at the time of sampling. In contrast, transmission intensity was medium-to-high in Chonyi (109 children, 1- to 10-years old, who were all asymptomatically infected at the time of blood sampling). In Ngerenya, 48% had antibodies mediating phagocytosis. This proportion rose to 90% among those who had asymptomatic infections at the time of sampling (Figure 5A), suggesting that active infection boosted opsonic antibodies. In Chonyi, where all children had asymptomatic infections, the prevalence of phagocytosis mediating antibodies was comparably high at 94.5% (Figure 5A). In both cohorts, the OPA activity positively correlated with age, reflecting increasing cumulative exposure to P. falciparum; this was statistically significant in the Ngerenya cohort (Figure 5B), but not in Chonyi (Figure 5C). In Ngerenya, the RPI was significantly higher in children who had active P. falciparum infections at sampling compared to aparasitemic children (Figure 5D). Opsonic phagocytosis was also higher for children who had P. falciparum infections in the six months prior to sample collection compared to those who had not been infected (mean RPI 33.4 versus 14.1, P
